Aquaporin-1 and HCO3(-)-Cl- transporter-mediated transport of CO2 across the human erythrocyte membrane

Abstract

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO3(-)-Cl- transporter may be involved in CO2 transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO2 transport in human red blood cell ghosts at physiological temperatures (37 degrees C). Replacement of inert with CO2-containing gas above a stirred cell suspension caused an outside-to-inside directed CO2 gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO2 passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO2-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl2 at 65 microM) or the HCO3(-)-Cl- transporter (by DIDS at 15 microM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO2, nearly the entire CO2 transport across the red cell membrane is mediated by AQP1 and the HCO3--Cl- transporter. Therefore, these proteins may function as high affinity sites for CO2 transport across the erythrocyte membrane.

Figure 1. Schematic diagram illustrating possible routes of CO₂ entry into the human erythrocyte and secondary transport processes

CO₂ may enter the erythrocyte either by directly diffusing through the lipid bilayer or by passing through membrane proteins (aquaporin-1, HCO₃⁻–Cl⁻ transporter) which would function as gas channels. Once CO₂ has entered the erythrocyte, it will be hydrated by CAII bound to the carboxyl terminus of the HCO₃⁻–Cl⁻ transporter. Intracellular carbonic acid then dissociates into a proton and bicarbonate. Both reaction products can be eliminated from the intracellular space by specific transporters, the Na⁺-H⁺ exchanger and the HCO₃⁻–Cl⁻ transporter. These processes will further increase the entry of CO₂ and therefore intracellular acidification. Net proton fluxes may also be linked to anion exchange activity (unspecified anion exchange with substrates A₁⁻ and A₂⁻).

Figure 2. Asymmetry of acidification and pH_i recovery

A, intracellular acidification in ghosts under exposure to carbogen. After 30 min, carbogen was replaced by N₂. The initial pH_i was regained after ∼90 min gassing with N₂. The rate of acidification is greater than the rate of pH_i recovery. B, recording of an acidification of ghost free buffer (10 mM Tris, pH 6.8). At this pH there is no buffer capacity left in a Tris–HCl buffer system. Therefore the steady state was reached after ∼30 min and pH is down to 6.1. Replacement of CO₂ with N₂ also showed an asymmetric pH recovery.

Figure 3. Steady state of pH_i after prolonged exposure to CO₂

Gassing a ghost suspension in PBS with carbogen (control) showed an initial fast acidification and then a slow approach to the physicochemical equilibrium, which was reached after ∼60 min. Addition of ETX (10 μM) to the ghost suspension blocked the initial fast component. The physicochemical equilibrium was not affected by the addition of ETX.

Figure 4. BCECF calibration curve and dependency of acidification rates on the concentration of CO₂

A, BCECF calibration curve measured in a ghost suspension at 1 μM nigericin in 10 mM phosphate buffered potassium chloride (154 mM, 300 mosmol kg⁻¹, pH 7.4 ± 0.15). Data were fitted to a sigmoidal mechanism (y = a + b/(1+e^{(-(x - c)/d)})), with r² > 0.999. Filled circles: pH_o, measured; continuous line: fit results; dashed lines: 95 % confidence. B, recordings of acidification in ghost free buffer with ∼1, 2, 3, 4 and 5 % CO₂ in the feeding gas. Values corrected for density differences were 0.884, 1.82, 2.81, 3.87, and 5 % CO₂, respectively. C, rates of acidification were obtained by fitting the recordings of acidification (B) to a mono exponential mechanism. Filled circles: calculated velocity constants (k) of a mono-exponential fit; continuous line: linear regression curve (r² > 0.975).

Figure 5. General protocol of an intracellular acidification of a ghost suspension under exposure to CO₂

Acidification was measured with the fluorescent dye BCECF. Excitation wavelengths were set to 502 and 440 nm, emission to 530 nm. The samples (1.5 ml) were incubated in fluorescence cells for 10 min, at 37 °C. Gas flow through a stopper was 2.5 l h⁻¹. The suspension was stirred at 950 r.p.m. First, inert gas (N₂) was used (open bars); then after an appropriate time (here 5 min), gas supply was switched to acidifying gas (carbogen, grey bar). If necessary the cycle was repeated. The graph can be fitted by several mono-exponential functions. a denotes the fast process of acidification, b the slow approach to physicochemical equilibrium.

Figure 6. Influence of the proton net back flux capacity on the extent of the fast acidification process

Comparison of the uncatalysed acidification (A) with a control assay (B) and experiments with retarded back flux (C and D). The samples were incubated under conditions as described in Fig. 5. A, a typical uncatalysed reaction. An uncatalysed reaction is achieved either by adding ETX (10 μM) to the ghost suspension or by gassing PBS to which BCECF (free acid) had been added (see Fig. 7A and B). B, a control experiment, which is the time course of acidification according to the standard protocol. C, a retarded back flux under conditions that inhibit the Na⁺-H⁺ antiporter (10 μM EIPA, incubated for 30 min prior to acidification). The arrow indicates the more pronounced plateau phase (kinetic data of acidification using a mono-exponential fit, see Table 1). D demonstrates that a temperature-sensitive transport mechanism is involved in the back flux of acidic equivalents. The temperature was lowered to 25 °C and caused a significant drop in acidification after the onset of CO₂, since the elimination of acidic equivalents is retarded (kinetic data of acidification using a mono-exponential fit, see Table 1).

Figure 7. Gassing of ghost-free buffer and gassing CAII-blocked ghost suspensions reveal identical time courses

Approach to the physicochemical equilibrium: comparison of the time course of acidification of uncatalysed reactions. A, time course of a ghost-free buffer (PBS: 10 mM, 300 mosmol kg⁻¹, pH: 7.4) solution to which BCECF (free acid) has been added. B, typical time course of acidification with blocked CAII activity through addition of the membrane-permeant ETX (10 μM). The time constants of both assays were not statistically different.

Figure 8. CAII activity in a ghost preparation is not rate limiting under experimental conditions

A, under the condition of resealing, ghosts were incubated with different amounts of human CAII (0, 200, 400 and 800 μl of 2.5 mg ml⁻¹ CAII). a denotes the fast acidification for the four assays. The statistical error of the initial rates is < 5 %, i.e. CAII load of a ghost preparation does not increase the rate or the ΔpH_i of the fast acidification. B, the rate of acidification after addition of CAII (50 μl) to a ghost suspension was very similar to the rate of acidification calculated for ghosts that had undergone lysis by a 1:6 dilution into hyposmotic (20 mosmol kg⁻¹) PBS.

Figure 9. Intracellular CAII is required for fast and biphasic acidification

Addition of CAII (50 μl of 2.0 mg ml⁻¹ human CAII) to the extracellular compartment of a ghost suspension (C) yields higher rates than the uncatalysed approach to the physicochemical equilibrium (A) (by addition of 10 μM ETX to the ghost suspension). No difference from Fig. 6A and Fig. 7B regarding the velocity constants could be detected. B, a control experiment with a fast (a) and a slow (b) acidification response.

Figure 10. Comparison of the effect of the AQP1 blocker pCMBS and HgCl₂

A, a control experiment. B, the sample has been incubated for 15 min with pCMBS at a final concentration of 1 mM. The change of pH in the fast phase is less and the time constant of that process is smaller compared to control. C represents the time course of an acidifying process measured in ghosts with HgCl₂ at a concentration of 62.5 μM. After onset of CO₂ the time course is not distinguishable from an uncatalysed reaction.

Figure 11. Establishment of a dose–response curve for the AQP1 inhibitor HgCl₂

A dose–response curve has been derived from acidification experiments in the presence of various concentrations of HgCl₂. Incubation time was 13 min. A, control with vehicle (PBS) added. B, time course at 10 μM HgCl₂. C, time course at 45 μM HgCl₂ and D, at 118 μM HgCl₂. The IC₅₀ calculated from the velocity constants was 39 ± 6 μM, n = 16.

Figure 12. Establishment of a dose–response curve for the HCO₃⁻–Cl⁻ transport inhibitor DIDS

A dose–response curve has been derived from acidification experiments in the presence of various concentrations of DIDS. The time course of acidification was registered after incubation with DIDS for 10 min. A, control with vehicle (PBS) added. B, time course at 1.5 μM DIDS. C, time course in the presence of 7.5 μM DIDS and D, at 15 μM DIDS. The IC₅₀ calculated from the velocity constants was 5.6 ± 1.2 μM, n = 25.

Figure 13. Comparison of acidification in the intracellular and extracellular compartment in control and inhibition experiments

Fluorescence inside the cell was measured as usual on BCECF-loaded cells. To measure outside the cells, ghosts were used that were not loaded with dye and added membrane-impermeant BCECF (free acid) to the outer compartment. A, control experiment (vehicle: PBS). B, same as A but with BCECF outside the cells. C, a, HgCl₂ (10 μM) was added prior to DIDS (8 μM); b, DIDS (8 μM) was added prior to HgCl₂ (10 μM). D, same as C a, but with BCECF outside the cells. E, DIDS (10 μM) present in the preparation, incubation 10 min. F, same as E, but with BCECF outside the cells.